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. 2023 May 10;13(1):6961.
doi: 10.1038/s41598-023-33893-7.

A system that delivers an antioxidant to mitochondria for the treatment of drug-induced liver injury

Mitsue Hibino  1   2 , Masatoshi Maeki  2 , Manabu Tokeshi  2 , Yoichi Ishitsuka  3 , Hideyoshi Harashima  1 , Yuma Yamada  4   5
Affiliations

A system that delivers an antioxidant to mitochondria for the treatment of drug-induced liver injury

Mitsue Hibino et al. Sci Rep. .

Abstract

Mitochondria, a major source of reactive oxygen species (ROS), are intimately involved in the response to oxidative stress in the body. The production of excessive ROS affects the balance between oxidative responses and antioxidant defense mechanisms thus perturbing mitochondrial function eventually leading to tissue injury. Therefore, antioxidant therapies that target mitochondria can be used to treat such diseases and improve general health. This study reports on an attempt to establish a system for delivering an antioxidant molecule coenzyme Q10 (CoQ10) to mitochondria and the validation of its therapeutic efficacy in a model of acetaminophen (APAP) liver injury caused by oxidative stress in mitochondria. A CoQ10-MITO-Porter, a mitochondrial targeting lipid nanoparticle (LNP) containing encapsulated CoQ10, was prepared using a microfluidic device. It was essential to include polyethylene glycol (PEG) in the lipid composition of this LNP to ensure stability of the CoQ10, since it is relatively insoluble in water. Based on transmission electron microscope (TEM) observations and small angle X-ray scattering (SAXS) measurements, the CoQ10-MITO-Porter was estimated to be a 50 nm spherical particle without a regular layer structure. The use of the CoQ10-MITO-Porter improved liver function and reduced tissue injury, suggesting that it exerted a therapeutic effect on APAP liver injury.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1
Schematic image of concept (A) of preparing CoQ10-MITO-Porter and (B) therapeutic strategy against APAP liver injury model using CoQ10-MITO-Porter. As shown in the panel (A), the iLiNP device contains the basic structure with 10 repetitions of baffle mixer structure. Standard dimensions of the baffle mixer structure were width (a) of 150 µm, depth (b) of 100 µm and interval (c) of 100 µm. APAP acetaminophen, CoQ10 coenzyme Q10, PEG polyethylene glycol, ROS reactive oxygen species, R8 octaarginine.
Figure 2
Figure 2
Evaluation of physical properties of the MITO-Porter with or without PEG, R8 and CoQ10. The prepared LNPs were characterized by three indices: (A) particle size, (B) dispersibility, (C) ζ-potential. The results of the physical properties of (a) Empty-MITO-Porter and (b) CoQ10-MITO-Porter are compared to consider the effect of CoQ10, a poorly water-soluble molecule. Circles represent the values of 4 individual samples and bars are the mean (n = 4). Data represented the mean ± S.D. (n = 4). Significant differences were calculated by two-way ANOVA, followed by Tukey test (***p < 0.001). In supplementary information, Table S1 shows the physical properties of the MITO-Porters.
Figure 3
Figure 3
Evaluation of the appearance of the MITO-Porter solution with or without PEG, R8 and CoQ10. (A) Turbidity of LNP solution. Data represent the mean ± S.D. (n = 4). The significant differences are calculated by two-way ANOVA, followed by Tukey test (***p < 0.001). (B) Appearance of LNP solution. (a) Empty-MITO-Porter and (b) CoQ10-MITO-Porter are compared. Table S2 shows the physical properties of the MITO-Porters.
Figure 4
Figure 4
Evaluation of the internal structure of CoQ10-MITO-Porter particles. (A) TEM image of CoQ10-MITO-Porter. Scale bar 50 nm. (B) SAXS profiles of LNPs. Red and blue dot plots show (A,B), respectively. (C) Conceivable fine structure of the CoQ10-MITO-Porter. Table S5 shows the physical properties of the MITO-Porters used for SAXS measurements.
Figure 5
Figure 5
Biodistribution of the CoQ10-MITO-Porter in mice. Ex vivo images of organs are obtained from non-treated or APAP-treated mice 3 h after injection with DiD-labeled CoQ10-MITO-Porter. (A) Amount transferred to several main organs. Based on the observed images, fluorescence intensity is quantified using image J and defined as the amount of transferred to the organs. Data represent the mean ± S.D. (n = 3). Significant differences were calculated by the Unpaired t-test (**p < 0.01). (B) Transfer rate on several main organs. The transfer rate is calculated from the transfer amount results. Data represent the mean ± S.D. (n = 3). The significant differences (vs Liver) were calculated by one-way ANOVA, followed by SNK test (**p < 0.01). (C) Biodistribution image on several main organs. (a) Non-treatment. (b) APAP-treatment. Table S6 shows the physical properties of the administered CoQ10-MITO-Porter.
Figure 6
Figure 6
Confirmation that the delivery of CoQ10 using the MITO-Porter system protects against APAP-induced liver injury. Schematic diagrams show (A) Time course for evaluating the therapeutic effect for APAP liver injury animal studies. Mice were injected with 200 mg/kg APAP intraperitoneally and after 1 h, with CoQ10-MITO-Porter and PBS (−) intravenously or CoQ10 suspension intraperitoneally. The CoQ10 dose was kept evenly at 0.9 mg/kg. The blood and liver from each mouse were collected at 24 h later from APAP-treatment. (B) Serum ALT levels. Data represent the mean ± S.D. (n = 3–6). Figure S3 showed serum LDH. Figure S4 describes a control experiment in which the empty-MITO-Porter was used. (C) Percentage of necrotic area based on the HE-stained whole liver sections. Data are displayed as the mean ± S.D. (n = 6–10). Panel (D) show representative images of stained sections of the liver: (a) PBS (−), (b) CoQ10 suspension, (c) CoQ10-MITO-Porter group. In HE-stained sections, dashed line encloses necrotic area. Scale bars are 100 µm. In TUNEL-stained sections, scale bar is 1000 µm. The significant differences (vs CoQ10-MITO-Porter group) were calculated by one-way ANOVA, followed by SNK test (**p < 0.01). The physical properties of the administered CoQ10-MITO-Porter are reported Table S7 in supplementary information.
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